The objective of this proposal is to understand the mechanism by which the protein, Sonic hedgehog (Shh), acts in a concentration-dependent manner to regulate retinal ganglion cell axon growth and guidance. Specifically, the receptor(s) to which Shh binds will be investigated to determine if the response to both low and high concentrations of Shh is mediated by one receptor or two different receptors, which has been reported for the concentration-dependent response of axons to the guidance factor, Wnt3. A co- immunoprecipitation experiment will be performed using tagged Shh protein to identify all candidate receptors to which Shh binds on axons. Expression of candidate receptors on axons will be investigated followed by functional studies to determine if the receptorsare required for the growth response of axons to Shh. Proteins that interact with the Shh signaling protein, Smoothened (Smo), will also be investigated. It has been shown that Smo is required for transducing the axon growth responses at both low and high concentrations and Smo binding proteins are required for transducing the hedgehog signal in other systems. To identify binding partners in axons, co-immunoprecipitation experiments using an anti-Smo antibody and a yeast two-hybrid screen using the cytoplasmic tail of Smo as bait will be performed. Examining Shh signaling at both the receptor and downstreamsignaling levels will lead to a better understanding of the molecular mechanisms by which Shh affects axon growth in a concentration-dependent manner and, more generally, how axon guidance factors may direct axongrowth. Overall, the proposed research relates to the mission of the NINDS in that it will lead to a better understanding of how the neural circuitry is established during development, a process that is critical for all neurological functions. Also, it may aid in the development of therapies for spinal cord injuries, in which axons are irreparably damaged, and lead to a better understanding of neurological disorders that may result from abnormal circuit formation, including epilepsy and autism. Understanding the mechanisms that regulate axon growth during development will be important in developing therapies for spinal cord injuries, in which nerve connections are damaged and for which there is currently no cure. Also, it has been suggested that some neurological disorders, including autism and epilepsy, may result from abnormal circuit formation during brain development. Therefore, researching how axons grow will be critical for identifying the causes of some neurological disorders and in developing a cure for people with debilitating spinal cord injuries.